The production of recrystallized S-layers from crystalline cell surface layers containing proteins (bacterial cell surface layers) on solid or liquid substrates is described, for example, in European Patent 0 154 620 B1 (=U.S. Pat. No. 4,752,395), which specifically relates to the production of multi-layer S-layers of prokaryotes and their use as ultra-filtration membranes.
S-layers are comprised of proteins that occur in prokaryotes as cell wall components, the amino acid sequence of an S-layer protein being generally type-specific or strain-specific; the S-layer proteins are usually simple proteins or glycoproteins. Currently, several hundred bacteria strains are known whose cell surfaces have crystalline structures. The lattice constants of these structures are within the range of approximately 3 to 35 nm, and the S-layers as monomolecular layers for the most part are 3 to 30 nm thick.
Isolated—split off from the original cell—S-layer proteins show the capability of assembling from a solution monomolecular crystal lattices (S-layers), which generally correspond to the crystal structures in the cell wall of an intact cell. Generally, a complete disintegration of S-layers in concentrated solutions of chaotropic agents is achievable by either lowering or raising the pH value. In the reconstitution of the S-layer proteins (“recrystallization”), flat layers, tubular structures such as cylinders and enclosed structures (vesicles) can form—so-called self-assembly products—and specifically as a function of the intrinsic properties of the S-layer proteins and the conditions in the S-layer formation, such as pH value, ion strength and ion composition of the solution; multi-layer S-layers can also form in addition to mono-layers. The formation of the S-layers occurs by self-assembly, because all information for the formation of the S-layer structure is contained in the individual monomers. The S-layers preferably form on boundary layers, for example, on the air/water boundary, on lipid films or on a solid substrate surface such as that of a silicon wafer, an electrode or a synthetic polymer. Due to the specific surface properties of the S-layer protein units, such as charge distribution, hydrophobia, specific interaction with components of the boundary layer, a defined orientation of the S-layer units (monomers, oligomers or small crystallites) occurs in the addition to the boundary layer and incorporation into the S-layer lattice.
S-layers represent highly porous membranes, wherein the pores as a part of the S-layer lattice have a uniform size and shape within the range of approximately 2-6 nm; the porosity can constitute up to 70% of the area. Using S-layer lattices for the immobilization of different molecules, because of the great thickness and defined position and orientation of functional groups that are arranged on the surface of the S-layer units, is known. In order to produce covalent bonds between the S-layer subunits and improve the stability characteristics, S-layer lattices were cross-linked with homobifunctional, amino-group-specific cross-linking agents of various lengths (for example, glutaraldehyde). Many enzymes that were immobilized on activated terminal groups of the S-layer proteins formed a monolayer of tightly arranged molecules on the outer surface of the S-layer lattice. The very specific morphological and biophysical characteristics of S-layer proteins, especially their capability of organizing themselves into monomolecular crystalline lattices that have on their surface a spatially defined arrangement of functional groups and pores of equal sizes and morphology, have led to a broad spectrum of applications in biotechnology, nanotechnology and biometrics.
For further details on S-layers, refer to the article, “Crystalline Bacterial Cell Surface Layers (S-Layers): A Versatile Self-assembly System” by U. B. Sleytr et al., Chapter 5 from “Supramolecular Polymers”, ed. A Ciferri, Marcel Dekker Inc., New York 2000 (ISBN 0-8247-0252-2).
A use of S-layers for the production of sensor systems is described by A. Neubauer et al. in Sensors and Actuators B 40 (1997) 231-236. This article deals with deposition of a gold layer on an S-layer by means of the so-called PLD method (“pulse laser deposition”, Deposition Using a Pulsed Laser under a High Vacuum Conditions), enzymes such as glucose oxidase having been immobilized on the S-layer beforehand. Additional methods for the deposition of metal layers on S-layers are addressed by, among others, D. Pum et al. in Ber. Bunsenges. Phys. Chem. 101 (1997) 1686-1689 and A. Neubauer et al. in PTB reports P-34 (1998), pp. 75-81. The latter article also discusses the electrochemical deposition of a metal layer (e.g. gold) on an S-layer but with less satisfactory success, because the deposited metal layer has a granular structure that does not correspond to the structure of the S-layer situated beneath it.
In the deposition of an S-layer from a solution that contains S-layer proteins, the S-layer units are deposited directly onto the substrate surface. They are first present unorganized on the surface; the formation of the crystalline structure occurs first in the course of the deposition process, starting in the usual way from crystallization nuclei. Especially if the density of the S-layer proteins on the substrate surface is not yet very large, the individual units can move around on the surface, the mobility being a function of, among other things, the type of the surface. The transition into an ordered structure frequently occurs spontaneously, if S-layer units are deposited in sufficient quantity on the substrate surface. Even after the formation of a crystalline S-layer, dissolved S-layer proteins can precipitate from the solution and attach to the S-layer lattice.
Already in known deposition methods, S-layer units in the solution assemble into S-layers and S-layer self-assembly products of undefined size that remain in solution or suspension or deposit onto the substrate. This operation thus enters into competition with the direct deposition of S-layer units onto the substrate. As a result, controlling the production process of an S-layer turns out to be difficult—especially if a mono layer is to be reliably deposited. Furthermore, the process of S-layer-unit deposition can result in the formation of crystalline double and multiple layers. This process is very disruptive for many applications, because the second (exposed) S-layer is for the most part bonded as a mirror image to the first S-layer (bonded to the substrate surface) and thus masks its functional S-layer domains that are needed for the application.
Moreover, the known methods are in need of improvement with regard to the long-trajectory ordering of the produced S-layer structures. Especially in the crystalline structures that are produced via the aforementioned method, the S-layer structures inevitably contain multiple domains whose expansion is thus rather small. In order to obtain domains of large area, it is desirable to limit the number of domains or the number of nuclei at which point the formation of the crystal structure of the S-layer starts.
European Patent 463 859 A2 describes the deposition of a biomolecular species (e.g. a protein such as glucose oxidase) on a biosensor electrode. In this context, starting from a solution in which the biomolecular species have the same electrical charge signs, a constant current is applied between the biosensor electrode and a counterelectrode, so that the biomolecules migrate between these electrodes to the biosensor electrode and collect there as a film. This publication is thus based on a galvanostatic method in which no consideration is given to a crystal-like ordering of the deposited biomolecules; moreover, the deposited layers, at approximately 1 μm or more, are relatively thick—unlike the actual S-layers in which one or a few (crytalline) monolayers are produced, each of which is 4 to 15 nm thick.
It is therefore the object of the present invention to be able to control the formation of an S-layer and the formation of the S-layer crystal structure in a manner that is an improvement over the known method.